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Acute neonatal hypoxia led to a gender-dependent upregulation of HIF1-α, GPx4 and BDNF mRNA expression level in the brain.Disturbance in the glutathione antioxidant system was revealed after acute neonatal hypoxia in blood and brain.Acute neonatal hypoxia induced a gender-dependent increase of BDNF protein level in the brain.A delay in physical and sensorimotor development of rat pups was observed in a gender-dependent manner.The model of acute neonatal hypoxia could be used as a milder model for hypoxic brain damage in extremely preterm infants.Perinatal hypoxia–ischaemia is one of the leading factors that negatively influence the development of the central nervous system. Our aim was to investigate the effects of sex on the outcomes of acute neonatal hypoxia (ANH) in rat pups. Male and female Wistar rats were exposed to a hypoxic condition (8% oxygen for 120min) at postnatal day 2 (P2). Immediately after ANH an increase in HIF1-α gene expression was observed in the rat brains, independently of sex. Brain-derived neurotrophic factor (BDNF) and glutathione peroxidase-4 gene expression was increased in female animals only. Hypoxic pups of both sexes showed a decreased reduced/oxidised glutathione (GSH/GSSG) ratio in the blood and only males had an increased GSH content in the whole brain immediately after hypoxia. Furthermore, an increased BDNF content in the brain was found in both male and female rat pups at 0h and in serum 4h after hypoxia, but at 4h after hypoxia only males had an increased BDNF level in the brain. Only hypoxic males displayed retarded performance in the righting reflex, but in a negative geotaxis test hypoxic pups of both sexes had an increased turnaround time. Moreover, hypoxic female but not male pups demonstrated less weight gain than control littermates for the entire observation period (until P18). These results demonstrate that ANH at P2 leads to both molecular and physiological impairments in a sex-specific manner and the described model could be used to represent mild hypoxic brain damage in very preterm infants.